A vast majority of biologically active molecules including growth factors, cytokines, neurotransmitters and hormones transduce signals via specific cell-surface receptors. Some of these receptors are then coupled to heterotrimeric GTP-binding proteins (G proteins) which, upon activation, relay signals to a variety of cellular effectors including at least four phospholipase C (PLC) variants and adenylyl cyclases.
G proteins mediate external signals by forming heterotrimers consisting of an alpha, beta and gamma subunit. Several isoforms of each subunit have been identified and therefore, through subunit heterogeneity, G proteins effectively integrate multiple signaling cascades.
The alpha subunits of G proteins contain the GTP binding site and intrinsic catalytic GTPase activity. Based on sequence similarity and function, these subunits have been classified into four major groups; Gs, which stimulate adenylyl cyclases; Gi, which inhibit adenylyl cyclases; Gq, which activate PLC isoforms and G12/13, which mediate pathways associated with cell growth and differentiation (Hamm, J. Biol. Chem., 1998, 273, 669-672).
G-alpha-16 is a member of the Gq subfamily of G proteins and was originally described as a protein that is expressed exclusively in hematopoietic cell lines (Amatruda et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 5587-5591). Since that time, G-alpha-16 mRNA has been identified in lymphoid cells, T-cells, human thymocytes and in platelets where the protein is localized predominantly in the cytosol and secretory granules (Giesberts et al., Biochem. Biophys. Res. Commun., 1997, 234, 439-444). Characterization of G-alpha-16 activity in Sf9 cells showed that the protein activates PLC-.beta.1, PLC-.beta.2 and PLC-.beta.3 but not PLC-.gamma.1 or PLC-.delta.1 (Kozasa et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 9176-9180).
G-alpha-16 has been shown to be quite non-selective, interacting differentially with several receptor types including members of the opioid and chemokine receptor families (Arai and Charo, J. Biol. Chem., 1996, 271, 21814-21819; Lee et al., J. Neurochem., 1998, 70, 2203-2211).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of G-alpha-16. To date, strategies aimed at inhibiting or investigating G-alpha-16 function have involved the use of constitutively active mutants of G-alpha-16 and antisense expression vectors.
Expression of a constitutively active form of G-alpha-16 (G.alpha.16Q212L) in vascular smooth muscle cells was shown to mimic the effects of vasoconstrictors on hypertrophy and muscle-specific gene expression (Higashita et al., J. Biol. Chem., 1997, 272, 25845-25850).
Antisense vector-mediated inhibition of G-alpha-16 has been utilized as a tool to elucidate G-alpha-16 mechanisms of signal transduction. Using antisense vectors expressing human G-alpha-16 cDNA in antisense orientation, Lippert et al. demonstrated that G-alpha-16 expression was tightly regulated during T-cell activation (Lippert et al., FEBS Lett., 1997, 417, 292-296). Other studies demonstrated that expression of full-length antisense to G-alpha-16 in human erythroleukemia cells resulted in the inability of the P2U (P2Y2) purinoceptor to stimulate the release of intracellular calcium suggesting that this receptor functions in a G-alpha-16 coupled pathway (Baltensperger and Porzig, J. Biol. Chem., 1997, 272, 10151-10159).
However, these strategies are untested as therapeutic protocols. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting G-alpha-16 function. Therefore, antisense oligonucleotides may provide a promising new pharmaceutical tool for the effective and specific modulation of G-alpha-16 expression.